Dear Netters,
about one month ago, I posted this message on behalf of a friend of mine:
> I am doing some assays that involve normal human AB+ serum. I test the serum
> for classical and alternative pathway activity before storing it at - 70 C.
> When the serum is thawed and the C5a levels measured, they are very low.
> However, after incubation at 37 C in sterile freezing vials or 96 well tissue
> culture plates the levels of C5a rise to comparable amounts found in serum
> activated by immune complexes. Since the experiments are concerned with the
> interactions between complement, immune complexes and various cells, the fact
> that the plastic itself appears to cause complement activation could be a
> problem.
> Has anyone else come across a similar problem? Are there any 96 well sterile
> plates which are known not to activate serum, or any coatings that might help?
We'd like to thank all the people who replied; following is the summary of the
answers we got.
Best regards, Luca.
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From: <U12201 at UICVM.CC.UIC.EDU>
Date: Sat, 18 Mar 1995 17:50:37 CST
To: <lj1 at mrc-lmb.cam.ac.uk> (Luca Jovine)
Subject: Re: Complement and plastic microtiters
Here is my resounding "perhaps" - I have no hands-on experince etc.
But my theory is that the activation is due to the hydrophobic nature
of the plates. I do know that Pierce (for brits: Pierce & Warriner)
has hydrophilic plates (their prod # is 15100) - here about $80 for a
pack of 5 ea. It is worth a try I would venture.
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Date: Mon, 20 Mar 1995 07:39:34 -0500
From: ranell at aol.com
Posted-Date: Mon, 20 Mar 1995 07:39:34 -0500
Reply-To: ranell at aol.com (Ranell)
To: lj1 at mrc-lmb.cam.ac.uk (Luca Jovine)
Subject: Re: Complement and plastic microtiters
X-Organization: America Online, Inc. (1-800-827-6364)
The person to contact , and referred to me as the man who wrote the book
on complement is a DR Michael Glovsky in Pasadena, California. I know of
him from taking my own children re: a complement deficiency. If you want
the # I can get it for you. E-mail me Ranell at aol.com
Ta Ta.
Ranell
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Date: Tue, 21 Mar 1995 16:45:33 -0600
From: herro001 at maroon.tc.umn.edu (Mike Herron)
To: lj1 at mrc-lmb.cam.ac.uk (Luca Jovine)
Subject: Re: Complement and plastic microtiters
Organization: U of MN, Dermatology
Yes, I did this in the early eighties. I am not sure if the activation is
due to the plastic or clotting factors left over from when the serum was
clotted or the plastic.
Corning/Costar makes a special plate (96 well and others) called "ultra
low binding plates". They do not bind proteins or cells so they might be
usefull here.
When I was doing the C5a RIAs in the 80s we collected the blood in
lavender vacutainer tubes with SOLID edta and then spun out the RBCs etc.
and added just enough calcium to neutralize the EDTA then incubated this
reconstituted plasma 20 minutes at 37 degrees C and wound the fibrin clot
onto a wooden stick to remove the clot (you gotta see this to believe it)
The we would precpitate the large protiens (leaving the C5a in solution)
and run the RIA. Alternatly we freeze the reconstituted serum and run the
RIA later.
Mike Herron
herro001 at maroon.tc.umn.edu
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Luca Jovine
Structural Studies Division
MRC Laboratory of Molecular Biology
Hills Road
Cambridge CB2 2QH
ENGLAND
Voice: +44.1223.402443
FAX: +44.1223.213556
Telex: 81532
E-Mail: lj1 at mrc-lmb.cam.ac.uk [lj1 at 131.111.84.16]
lj200 at cus.cam.ac.uk [lj200 at 131.111.8.1]
lj200 at hermes.cam.ac.uk [lj200 at 131.111.8.34]
jovinel at imiucca.csi.unimi.it [jovinel at 159.149.10.1]
"I'll give you a definitive maybe" (Sam Goldwyn)
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